A role for neuroimmune signaling in a rat model of Gulf War Illness-related pain

Abstract

More than a quarter of veterans of the 1990-1991 Persian Gulf War suffer from Gulf War Illness (GWI), a chronic, multi-symptom illness that commonly includes musculoskeletal pain. Exposure to a range of toxic chemicals, including sarin nerve agent, are a suspected root cause of GWI. Moreover, such chemical exposures induce a neuroinflammatory response in rodents, which has been linked to several GWI symptoms in rodents and veterans with GWI. To date, a neuroinflammatory basis for pain associated with GWI has not been investigated. Here, we evaluated development of nociceptive hypersensitivity in a model of GWI. Male Sprague Dawley rats were treated with corticosterone in the drinking water for 7 days, to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate. These exposures alone were insufficient to induce allodynia. However, an additional sub-threshold challenge (a single intramuscular injection of pH 4 saline) induced long-lasting, bilateral allodynia. Such allodynia was associated with elevation of markers for activated microglia/macrophages (CD11b) and astrocytes/satellite glia (GFAP) in the lumbar dorsal spinal cord and dorsal root ganglia (DRG). Additionally, Toll-like receptor 4 (TLR4) mRNA was elevated in the lumbar dorsal spinal cord, while IL-1β and IL-6 were elevated in the lumbar dorsal spinal cord, DRG, and gastrocnemius muscle. Demonstrating a casual role for such neuroinflammatory signaling, allodynia was reversed by treatment with either minocycline, the TLR4 inhibitor (+)-naltrexone, or IL-10 plasmid DNA. Together, these results point to a role for neuroinflammation in male rats in the model of musculoskeletal pain related to GWI. Therapies that alleviate persistent immune dysregulation may be a strategy to treat pain and other symptoms of GWI.

Keywords: Cytokines; Glia; Gulf War Illness; Immune cell; Neuroimmune; Pain; Sphingolipids.

Figure 1.. Timeline of experimental procedures.

CORT: corticosterone; DFP: diisopropyl fluorophosphate; Veh: vehicle.

Figure 2.. Mechanical allodynia in a putative model of GWI pain.

Corticosterone (CORT) or vehicle was administered via the drinking water for 7 days to mimic high physiological stress. On the final day of CORT treatment, a single dose of diisopropyl fluorophosphate (DFP) or vehicle was administered. Seven days later, rats received a single injection of normal (pH = 7.2) or acidic saline (pH = 4.0) into the left gastrocnemius muscle. (A) Treatment with CORT alone, DFP alone, or in combination (all following treatment with intramuscular normal saline) did not alter mechanical thresholds. (B) Acidic saline in combination with CORT and DFP significantly reduced mechanical thresholds. Relative to CORT+DFP+Normal saline: *P < 0.05, **P < 0.01, ***P < 0.001; Relative to Vehicle+Vehicle+Acidic saline: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; Relative to Vehicle+Vehicle+Normal saline: ^{^^}P < 0.01, ^{^^^}P < 0.001. N = 6–8/group. Data are mean ± SD.

Figure 3.. Neuroinflammatory markers in the lumbar dorsal spinal cord.

Corticosterone (CORT) or vehicle was administered via the drinking water for 7 days to mimic high physiological stress. On the final day of CORT treatment, a single dose of diisopropyl fluorophosphate (DFP) or vehicle was administered. Seven days later, rats received a single injection of normal (pH = 7.2) or acidic saline (pH = 4.0) into the left gastrocnemius muscle. Left L4/5 dorsal spinal cord quadrants were collected 5 or 15 days later. Expression levels of (A, B) Itgam (CD11b), (C, D) Gfap, and (E, F) Tlr4 were assessed. Protein levels of (G, H) IL-1β, and (I, J) IL-6 were also assayed. *P < 0.05, **P < 0.01, ***P < 0.001. N = 6–8/group. Data are mean ± SD.

Figure 4.. Levels of neuroinflammatory markers in DRG.

Corticosterone (CORT) or vehicle was administered via the drinking water for 7 days to mimic high physiological stress. On the final day of CORT treatment, a single dose of diisopropyl fluorophosphate (DFP) or vehicle was administered. Seven days later, rats received a single injection of normal (pH = 7.2) or acidic saline (pH = 4.0) into the left gastrocnemius muscle. Left L4/5 DRG were collected 5 or 15 days later. Expression levels of Itgam (CD11b) were determined at (A) day 5, (B) and day 15, as well as Gfap at (C) day 5, (D) and day 15. Protein levels of IL-1β were assayed at (E) day 5 (F) and day 15. IL-6 levels were also assayed at (G) day 5 (H) and day 15, using the lower limit of detection for statistical analysis of undetectable values. *P < 0.05, **P < 0.01, ***P < 0.001. N = 6–8/group. Data are mean ± SD.

Figure 5.. Pro-inflammatory cytokine levels in gastrocnemius muscle.

Corticosterone (CORT) or vehicle was administered via the drinking water for 7 days to mimic high physiological stress. On the final day of CORT treatment, a single dose of diisopropyl fluorophosphate (DFP) or vehicle was administered. Seven days later, rats received a single injection of normal (pH = 7.2) or acidic saline (pH = 4.0) into the left gastrocnemius muscle. Gastrocnemius muscle was collected 5 or 15 days later. (A) IL-1β levels were assayed at day 5, (B) and day 15. (C) IL-6 levels were assayed at day 5, (D) and day 15. *P < 0.05, **P < 0.01, ***P < 0.001. N = 6–8/group. Data are mean ± SD.

Figure 6.. Effects of immunomodulators on allodynia in a putative model of GWI pain.

Corticosterone (CORT) or vehicle was administered via the drinking water for 7 days to mimic high physiological stress. On the final day of CORT treatment, a single dose of diisopropyl fluorophosphate (DFP) or vehicle was administered. Seven days later, rats received a single injection of normal (pH = 7.2) or acidic saline (pH = 4.0) into the left gastrocnemius muscle. Allodynia was attenuated by (A) treatment with the macrophage/microglia inhibitor minocycline (50 mg/kg once per day), beginning on day 5 and concluding on day 12 (indicated by the gray rectangle), (B) the TLR4 inhibitor (+)-naltrexone (6 mg/kg three times per day), beginning on day 5 and concluding on day 12 (indicated by the gray rectangle), and (C) IL-10 gene therapy (3 μg of plasmid DNA with 25 μg D-mannose), intrathecally administered on day 3 (indicated by the arrow). *P < 0.05, **P < 0.01, ***P < 0.001. N = 6–8/group. Data are mean ± SD.